Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes

To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5). Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5). The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain. However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain. These results suggest the existence in L. monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase. sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L. monocytogenes. The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain. Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain. sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L. monocytogenes against environmental adversities.     

Relative levels and fractionation properties of Bacillus subtilis sigma(B) and its regulators during balanced growth and stress.

sigma B is a secondary sigma factor that controls the general stress response in Bacillus subtilis. sigma B-dependent genes are activated when sigma B is released from an inhibitory complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate pathways, responding either to a drop in intracellular ATP levels or to environmental stress (e.g., heat, ethanol, or salt), cause the release of sigma B from RsbW. rsbR, rsbS, rsbT, and rsbU are four genes now recognized as the upstream half of an operon that includes sigB (sigma B) and its principal regulators. Using reporter gene assays, we find that none of these four genes are essential for stationary-phase (i.e., ATP-dependent) activation of sigma B, but rsbU and one or more of the genes contained within an rsbR,S,T deletion are needed for stress induction of sigma B. In other experiments, Western blot (immunoblot) analyses showed that the levels of RsbR, RsbS, Rsb, and RsbU, unlike those of the sigB operon's four downstream gene products (RsbV, RsbW, RsbX and sigma B), are not elevated during sigma B activation. Gel filtration and immunoprecipitation studies did not reveal the formation of complexes between any of the four upstream sigB operon products and the products of the downstream half of the operon. Much of the detectable RsbR, RsbS, RsbT, and RsbU did, however, fractionate as a large-molecular-mass (approximately 600-kDa) aggregate which was excluded from our gel filtration matrix. The downstream sigB operon products were not present in this excluded material. The unaggregated RsbR, RsbS, and RsbU, which were retarded by the gel matrix, elated from the column earlier than expected from their molecular weights. The RsbR and RsbS fractionation profile was consistent with homodimers (60 and 30 kDa, respectively), while the RsbU appeared larger, suggesting a protein complex of approximately 90 to 100 kDa.     

Deletion of the sigB gene in Bacillus cereus ATCC 14579 leads to hydrogen peroxide hyperresistance

The sigB gene of Bacillus cereus ATCC 14579 encodes the alternative sigma factor sigma(B). Deletion of sigB in B. cereus leads to hyperresistance to hydrogen peroxide. The expression of katA, which encodes one of the catalases of B. cereus, is upregulated in the sigB deletion mutant, and this may contribute to the hydrogen peroxide-resistant phenotype.     

Reactivation of the Bacillus subtilis anti-sigma B antagonist, RsbV, by stress- or starvation-induced phosphatase activities.

sigma B is a secondary sigma factor that controls the general stress regulon in Bacillus subtilis. The regulon is activated when sigma B is released from a complex with an anti-sigma B protein (RsbW) and becomes free to associate with RNA polymerase. Two separate mechanisms cause sigma B release: an ATP-responsive mechanism that correlates with nutritional stress and an ATP-independent mechanism that responds to environmental insult (e.g., heat shock and ethanol treatment). ATP levels are thought to directly affect RsbW's binding preference. Low levels of ATP cause RsbW to release sigma B and bind to an alternative protein (RsbV), while high levels of ATP favor RsbW-sigma B complex formation and inactivation of RsbV by an RsbW-dependent phosphorylation. During growth, most of the RsbV is phosphorylated (RsbV-P) and inactive. Environmental stress induces the release of sigma B and the formation of the RsbW-RsbV complex, regardless of ATP levels. This pathway requires the products of additional genes encoded within the eight-gene operon (sigB) that includes the genes for sigma B, RsbW, and RsbV. By using isoelectric focusing techniques to distinguish RsbV from RsbV-P and chloramphenicol treatment or pulse-chase labeling to identify preexisting RsbV-P, we have now determined that stress induces the dephosphorylation of RsbV-P to reactivate RsbV. RsbV-P was also found to be dephosphorylated upon a drop in intracellular ATP levels. The stress-dependent and ATP-responsive dephosphorylations of RsbV-P differed in their requirements for the products of the first four genes (rsbR, -S, -T, and -U) of the sigB operon. Both dephosphorylation reactions required at least one of the genes included in a deletion that removed rsbR, -S, and -T; however, only an environmental insult required RsbU to reactivate RsbV.     

The rpoE operon regulates heat stress response in Burkholderia pseudomallei

Burkholderia pseudomallei is a gram-negative bacterium and the causative agent of melioidosis, one of the important lethal diseases in tropical regions. In this article, we demonstrate the crucial role of the B. pseudomallei rpoE locus in the response to heat stress. The rpoE operon knockout mutant exhibited growth retardation and reduced survival when exposed to a high temperature. Expression analysis using rpoH promoter-lacZ fusion revealed that heat stress induction of rpoH, which encodes heat shock sigma factor (sigma(H)), was abolished in the B. pseudomallei rpoE mutant. Analysis of the rpoH promoter region revealed sequences sharing high homology to the consensus sequence of sigma(E)-dependent promoters. Moreover, the putative heat-induced sigma(H)-regulated heat shock proteins (i.e. GroEL and HtpG) were also absent in the rpoE operon mutant. Altogether, our data suggest that the rpoE operon regulates B. pseudomallei heat stress response through the function of rpoH.     

Deletion of sigB in Bacillus cereus affects spore properties

In Bacillus cereus and other gram-positive bacteria the alternative sigma factor sigma(B) is an important regulator of the stress response. Deletion of the sigB gene generally leads to a stress-sensitive phenotype of vegetative cells. In this study, we describe the effect of the deletion of the sigB gene in B. cereus on spore properties. In particular, spores of the sigB deletion mutant showed a defect in germination upon exposure to the germinants alanine and inosine.     

Occurrence of mazEF-like antitoxin/toxin systems in bacteria

The mazEF locus of Escherichia coil located in an operon together with the upstream relA gene (encoding ATP:GTP 3'-pyrophosphotransferase; (p)ppGpp synthetase), encodes an antitoxin/toxin system which might play a role in programmed cell death under stress and starvation conditions at high cell densities. By homology searches, chromosomally encoded orthologous systems were identified in a variety of bacteria, sometimes without the MazE-like antitoxin, and several bacterial species possess multiple MazEF-like systems (paralogs). In many gram positive bacteria, the mazEF-locus is located directly upstream of the sigB (stress sigma factor sigmaB) operon in a putative operon together with the upstream dal (aIr) gene (encoding D-alanine racemase). The acidic antitoxins are less conserved than the basic toxins. The differences in genomic organization of the mazEFloci in E. coli versus those in gram positive bacteria might indicate their association with different stress response regulons in these organisms. A study on the sigmaB operon of Staphylococcus aureus showed that the mazF gene of this organism is cotranscribed with the sigmaB operon in response to heat shock, providing the first example that the expression of the mazEFlocus might be indeed associated with stress responses.     

The single extracytoplasmic-function sigma factor of Xylella fastidiosa is involved in the heat shock response and presents an unusual regulatory mechanism

Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.     

Regulation of sigma B levels and activity in Bacillus subtilis.

The sigB operon of Bacillus subtilis encodes sigma B plus three additional proteins (RsbV, RsbW, and RsbX) that regulate sigma B activity. Using an anti-sigma B monoclonal antibody to monitor the levels of sigma B protein, PSPAC to control the expression of the sigB operon, and a ctc-lacZ reporter system to monitor sigma B activity, we observed that the rsbV and rsbW products control sigma B activity at the ctc promoter independently of their effects on sigma B levels. In contrast, RsbX was found to have no effect on expression of ctc when the sigB operon was controlled by PSPAC. The data are consistent with RsbV and RsbW being regulators of sigma B activity and RsbX acting primarily as a negative regulator of sigB operon expression. Evidence that stationary-phase induction of the sigma B-dependent ctc promoter is accomplished by a reduction in RsbW-dependent inhibition of sigma B activity is also presented. In addition, Western blot (immunoblot) analyses of sigB operon expression demonstrated that sigma B accumulation is coupled to the synthesis of its primary inhibitor (RsbW). This finding is consistent with RsbW and sigma B being present within the cell in equivalent amounts, a circumstance that would permit RsbW to directly influence sigma B activity by a direct protein-protein interaction.     

Listeria monocytogenes sigma B regulates stress response and virulence functions

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.